The functional near-infrared spectroscopy (fNIRS) can detect hemodynamic responses in the brain and the data consist of bivariate time series of oxygenated hemoglobin (oxy-Hb) and deoxygenated hemoglobin (deoxy-Hb) on each channel. In this study, we investigate oscillatory changes in infant fNIRS signals by using the oscillator decompisition method (OSC-DECOMP), which is a statistical method for extracting oscillators from time series data based on Gaussian linear state space models. OSC-DECOMP provides a natural decomposition of fNIRS data into oscillation components in a data-driven manner and does not require the arbitrary selection of band-pass filters. We analyzed 18-ch fNIRS data (3 minutes) acquired from 21 sleeping 3-month-old infants. Five to seven oscillators were extracted on most channels, and their frequency distribution had three peaks in the vicinity of 0.01-0.1 Hz, 1.6-2.4 Hz and 3.6-4.4 Hz. The first peak was considered to reflect hemodynamic changes in response to the brain activity, and the phase difference between oxy-Hb and deoxy-Hb for the associated oscillators was at approximately 230 degrees. The second peak was attributed to cardiac pulse waves and mirroring noise. Although these oscillators have close frequencies, OSC-DECOMP can separate them through estimating their different projection patterns on oxy-Hb and deoxy-Hb. The third peak was regarded as the harmonic of the second peak. By comparing the Akaike Information Criterion (AIC) of two state space models, we determined that the time series of oxy-Hb and deoxy-Hb on each channel originate from common oscillatory activity. We also utilized the result of OSC-DECOMP to investigate the frequency-specific functional connectivity. Whereas the brain oscillator exhibited functional connectivity, the pulse waves and mirroring noise oscillators showed spatially homogeneous and independent changes. OSC-DECOMP is a promising tool for data-driven extraction of oscillation components from biological time series data. The functional near-infrared spectroscopy (fNIRS) can detect hemodynamic responses in the brain and the data consist of bivariate time series of oxygenated hemoglobin (oxy-Hb) and deoxygenated hemoglobin (deoxy-Hb) on each channel. In this study, we investigate oscillatory changes in fNIRS signals of infants by using a statistical method for extracting oscillators from time series data, which we call the oscillator decompisition method (OSC-DECOMP). OSC-DECOMP determines the number, frequencies and powers of oscillators as well as their projection patterns in a data-driven manner and does not require arbitrary selection of band-pass filters. Three types of oscillators  were found and each oscillator showed a characteristic spatial synchrony. Model comparison with the Akaike Information Criterion (AIC) demonstrated that the time series of oxy-Hb and deoxy-Hb on each channel originate from common oscillatory activity. We believe that OSC-DECOMP will become a promising tool of neural oscillation analysis for not only fNIRS but also LFP, EEG, MEG and fMRI. Spontaneous changes in cerebral oxygenation reflect hemodynamic response to spontaneous neural activity. The functional Near-Infrared Spectroscopy (fNIRS) has been used to detect the low frequency oscillations of cerebral oxygenation with frequency range between 0.01 and 0.1 Hz in adults , elders Recently, a statistical method for extracting oscillators from time series data was developed , 17, calOSC-DECOMP is also applicable to multivariate time series data . Fig 2 sIn this study, we investigated oscillatory changes in infant fNIRS data by using OSC-DECOMP. Specifically, we analyzed fNIRS data (3 minutes) acquired from sleeping 3-month-old infants  and extrIn the present study, we analyze data that was previously reported in , 18. TheWe analyzed the data obtained from 21 full-term infants  as they slept naturally.We used an fNIRS instrument  with 94 measurement channels (47 channels in each hemisphere) to detect the relative concentration changes in oxy-Hb and deoxy-Hb [millimolar-millimeter (mM ⋅ mm)] for 3 min without presenting external stimuli. The sampling rate was set at 10 Hz, resulting in 1,800 points per channel of spontaneous changes in oxy- and deoxy-Hb signals. Two sets of 3 × 10 array probes composed of 15 sources and 15 detectors of NIR light , which defined 47 channels, were mounted on a flexible cap over the frontal, temporal, parietal, and occipital regions of the left and right hemispheres. The distance between the source and detector was set to approximately 2.0 cm. The international 10/20 system of electrode placement was referenced to set the positions of the measurement channels. We previously estimated the locations of channels using a magnetic resonance imaging brain atlas . To evalhttp://www.stat.t.u-tokyo.ac.jp/~t-matsuda/software.html. Here, we briefly review this method. See To extract the underlying oscillators from the bivariate time series of oxy- and deoxy-Hb on each channel, we applied the oscillator decompisition method (OSC-DECOMP) , 17. They =   be a J-dimensional time series data of length N with sampling period Δt. The current fNIRS data corresponds to J = 2, N = 1800 and Δt = 0.1 s. OSC-DECOMP assumes that there are K oscillators underlying y, each of which is represented by a point on a two-dimensional plane that rotates around the origin with random fluctuation  is the argument of the complex number z  is modeled asyt,j is assumed to be the sum of the K inner products of the vectors  with the oscillator coordinates wt,j. Recall that the inner product A ⋅ B of two vectors A and B is given byA‖ is the length (norm) of A and ϕ is the angle between A and B. Therefore, the observation model   =  for k = 1, …, K.By using the  icjk,2) . In ordey1, …, yN are Gaussian and their mean and covariance can be computed by the Kalman smoother algorithm  [K = 1, …, Kmax and then select K byThe pair  and 2)  2) formson (AIC) . SpecifiJ = 2 to the bivariate time series of oxy- and deoxy-Hb on each channel. This model is based on the assumption that the two time series originate from common oscillators. Whereas this assumption seems reasonable for the fNIRS data, there is another possibility that each time series originates from its own oscillators and thus develops independently with each other. Namely, we can consider the state space model (J = 1 [We fitted the state space model  and 2)  2) with ce model  and (2) l ) and p(2)). Let θ(1) and θ(2), respectively. Then, the AIC of these models arez is approximately the standard normal N. Therefore, the p-value is calculated asp is smaller than the significance level α. For verification, we also applied the Wilcoxon signed-rank test to z.A statistical model for time series data t’s test , we consFinally, we investigated the frequency-specific functional connectivity based on the result of OSC-DECOMP.a, b) ≠ . This problem is reduced to the generalized eigenvalue problem:XX, ΣXY, ΣYX, ΣYY are submatrices of the covariance matrix of , given byr is regarded as a measure of the linear dependence between X and Y.As a result of OSC-DECOMP, we obtained a bivariate time series for each oscillator. To evaluate the correlation between two oscillators, we employed canonical correlation analysis, which is a statistical method for investigating the relationship between two sets of random variables. The (first) canonical correlation coefficient between two random vectors fk in the target frequency band on each channel. If there was more than one oscillator in the target frequency band, we selected the oscillator with the maximum power. Next, for each pair of channels, we calculated the (first) canonical correlation coefficient between the selected oscillators. Namely, we substituted the empirical covariance matrix of the oscillators into the preceding formula. We then considered the channel pairs with a canonical correlation coefficient that exceeded the threshold as functionally connected. Thus, we obtained a network of frequency-specific functional connectivity.To determine the functional connectivity for target frequency bands, we first selected an oscillator with a frequency Before presenting the results on real data, we first validate the performance of OSC-DECOMP on synthetic data with comparison to the conventional method with bandpass filtering. For more experiments, see , 17.J = 2, K = 3, N = 1800, Δt = 0.1s andWe generated bivariate time series from the state space model  and 2)  2) with bandpass with passband 0.01–0.1 Hz. The mean squared error in reconstructing the first oscillation component was 5.1 × 10−4 for OSC-DECOMP and 5.6 × 10−3 for bandpass filtering. Thus, OSC-DECOMP extracts the oscillation component more accurately than bandpass filtering.Conventionally, oscillation components in time series data are extracted by using the bandpass filters. Here, we compare the accuracy of OSC-DECOMP and bandpass filtering in reconstructing the first oscillation component (time series of the first coordinate of the first oscillator). Note that the second and third oscillators are difficult to separate by the bandpass filter because their spectra significantly overlap. For bandpass filtering, we used the MATLAB function We also compare the accuracy of OSC-DECOMP and bandpass filtering in estimating the phase difference between two time series (e.g. oxy- and deoxy-Hb). For the same reason as the previous paragraph, we focus on the phase difference for the first oscillator, which is 230°. For OSC-DECOMP, the estimate of the projection vector was ormation ) and theormation , the 95%ormation . See S1 21,2)=-0.5,-0.417 ormation . The resτ2 of observation noise. It demonstrates that the estimation accuracy of OSC-DECOMP is almost constant as long as τ2 is smaller than 10−3.To investigate the oscillatory changes in the 18-ch fNIRS data (3 minutes) taken from 21 sleeping 3-month-old infants, we applied OSC-DECOMP to the bivariate time series of oxy- and deoxy-Hb on each channel.εo(λ) and εd(λ) are the extinction coefficients of oxy- and deoxy-Hb at a given wave length λ, respectively, and L is the optical pathlength. However, the detected intensities of the two wavelengths of light include measurement noise in addition to changes due to absorption by chromophores asN(λ) is applied noise at a given wave length λ. Then, we obtain the following equation asE = 1/(εo(λ2)εd(λ1) − εo(λ1)εd(λ2))L. This shows that the measurement noise for each wavelength is transformed to noisy signals of oxy- and deoxy-Hb with opposite sign. Thus, the first type is referred to as a “pulse wave oscillator,” and the second type as a “mirroring noise oscillator.” ak ∈  specifies the width of the spectral peak at the frequency fk. Namely, the width of the spectral peak is smaller for ak closer to one. ak for each type of oscillators. It indicates that the spectral peak is very sharp for the brain oscillators and pulse wave oscillators. In other words, the waveform is close to sinusoidal for these oscillators. It implies that the brain oscillators cannot be interpreted as non-oscillatory drift, although its frequency is close to zero. On the other hand, for the mirroring noise oscillators, the parameter ak is concentrated around 0.4. Thus, the mirroring noise is considered to be non-sinusoidal.In the oscillator model , the parp < 10−16 in all cases. The Wilcoxon signed-rank test, which does not require the normality assumption on the test statistic, also demonstrated that these AIC differences were significant with p < 10−12 in more than 90% cases. Therefore, the assumption that common oscillators underlie the oxy- and deoxy-Hb time series is indeed supported by the experimental data. For comparison, we applied the same analysis to pairs of oxy- and deoxy-Hb data that were randomly shuffled across the subjects. The result is shown in To verify that the time series of oxy- and deoxy-Hb on each channel originate from common oscillatory activity, we compared the Akaike Information Criterion (AIC) of two state space models. p < 0.05; L5 and R8, p = 0.005) and mirroring noise . Finally, we examined the frequency-specific functional connectivity using the canonical correlation coefficients as explained in Material and Methods. We considered that this method revealed spatial synchrony when oscillators of pulse waves and mirroring noise were analysed. For each of the three oscillators shown in To evaluate the aforementioned visual inspections, we categorized channel pairs into the following four groups  based onFor comparison, Here, we compare OSC-DECOMP with the empirical mode decomposition (EMD) . EMD is EMD is an algorithm for decomposing univariate nonstationary time series data into several mode functions by focusing on the local properties of the signal. It does not aim at extracting common oscillators in multivariate time series data. On the other hand, OSC-DECOMP extracts oscillators from both univariate and multivariate stationary time series data based on statistical models. It is an interesting future work to extend OSC-DECOMP to nonstationary data.In this study, we investigated the oscillatory activity in infant fNIRS data by using the oscillator decompisition method (OSC-DECOMP) , 17. FivNeural oscillations are relevant in many brain functions . For exaIn contrast, OSC-DECOMP extracts the oscillation components in a data-driven (objective) manner using a statistical modeling approach. Both the number and frequencies of underlying oscillators are determined based on data. When applied to multivariate time series data, this method also estimates the projection pattern between each time series and each oscillator, which describes the amplitude and phase modulation. Thus, even if there are oscillators with very close frequencies, OSC-DECOMP can separate them correctly if their projection patterns are sufficiently different. The current result on pulse wave and mirroring noise can be interpreted as a typical example of this. Therefore, OSC-DECOMP enables the investigation of the common oscillators that underly multivariate time series in a data-driven manner. Several studies have focused on the phase difference between the band-pass filtered signals of oxy- and deoxy-Hb. For example,  investigThe present study based on OSC-DECOMP demonstrated that the brain, pulse wave, and mirroring noise oscillators can be extracted from the fNIRS signals obtained from the cranium of sleeping infants. In the time domain, fNIRS signals with slow components between 0.01 Hz and 0.1 Hz generally include hemodynamic and oxygenation changes in response to brain activity, similarly to fMRI –39. The For frequencies higher than 0.1 Hz, fNIRS signals include components associated with respiration (-0.3 Hz) and heartbeat (-1 Hz), in the case of adults . In the fNIRS signals are assumed to reflect changes in hemodynamics and oxygenation in cerebral tissue. However, those that originate from extracerebral tissue can have an impact on the fNIRS signals . In the In this study, we examined frequency-specific functional connectivity by calculating the canonical correlation between two oscillators in different channels Figs  and 18. The short-range and contralateral-transverse connectivity of the brain oscillators were higher than the ipsilateral-longitudinal connectivity and the control . These rIn the present study, we first determined the brain oscillators for each measurement channel. The functional connectivity was then evaluated by calculating the correlation between the time series of the oscillators. Another way of defining functional connectivity between channels is to determine whether the change in the oxy-Hb and deoxy-Hb signals in two or more channels is caused by common oscillatory activity. For example, each of the two channels measures a set of time series of oxy-Hb and deoxy-Hb signals, and a total of four time series can be used to investigate the presence of a common oscillator. If there is a low-frequency oscillator with power above a certain threshold, these two channels can be considered to exhibit significant functional connectivity. A functional network can also be defined by establishing whether there is a common oscillator among multiple channels. This method can be applied to time series of functional magnetic resonance imaging (fMRI), magnetoencephalography (MEG), electrocorticography (ECoG), and multi-electrode EEG to clarify functional relationships across broad brain regions.S1 AppendixThe supplementary material includes the technical details on (1) state space models and time series decomposition, (2) Kalman filter/smoother algorithm, and (3) Hessian computation and confidence interval.(PDF)Click here for additional data file.


Immune-related long non-coding RNAs (irlncRNAs) appear valuable in predicting prognosis in patients with cancer. In this study, we used a fresh modeling algorithm to construct irlncRNAs signature and then assessed its predictive value for prognosis, tumor immune infiltration, and chemotherapy efficacy in gastric cancer (GC) patients.The raw transcriptome data were extracted from the Cancer Genome Atlas (TCGA). Patients were randomly divided into the training and testing cohort. irlncRNAs were identified through co-expression analysis, after which differentially expressed irlncRNA (DEirlncRNA) pairs were identified. Next, we developed a model to distinguish between high- or low-risk groups in GC patients through univariate and LASSO regression analyses. A ROC curve was used to verify this model. After subgrouping patients according to the median risk score, we investigated the connection between the risk score of GC and clinicopathological characteristics. Functional enrichment analysis was also performed.We find that the results indicate that immune-related lncRNA signaling has essential value in predicting prognosis, and it may be potential to measure the Efficacy for immunotherapy. This feature may be a guide to the selection of GC immunotherapy.Our data revealed that immune-related lncRNA signaling had essential value in predicting prognosis, and it may be potentially used to measure the efficacy for immunotherapy. This feature may also be used to guide the selection of GC immunotherapy. Helicobacter pylori infection, age, high salt intake, and low intake of fruits and vegetables (Gastric cancer (GC) is the 5th most common neoplasm and the 3rd most deadly cancer worldwide –6. Althogetables , 8. At pOver the years, immunotherapy has been widely applied to treat patients with advanced gastric cancer. New strategies have been based on targeting or manipulating the immune system to reactivate the function of anti-tumor , 10. ConHuman transcriptome includes many non-coding RNAs, including long non-coding RNAs (lncRNAs) with a length of more than 200 nucleotides . These sThe accuracy of prognostic cancer models based on the combination of two biomarkers is superior to a single gene marker . In thishttps://portal.gdc.cancer.gov/repository), including 375 GC tissues and 32 non-tumor tissues. Immune-related genes (ir-genes) were obtained from the ImmPort database (http://www.immport.org). A co-expression strategy was used to identify irlncRNAs. The immune gene correlation coefficients above 0.4 and P-values < 0.001 were considered to be irlncRNAs.The data of RNA expression profiles and clinical features for GC were downloaded from The Cancer Genome Atlas (TCGA) program  and a test group (n = 150). Differential expression analysis between irlncRNAs performed using the R package Dseq2 identified the differentially expressed irlncRNA (DEirlncRNA) in the training cohort with log fold change |FC| > 2.0 and p-value < 0.05.Using the R package A 0-or-1 matrix was constructed by cyclically singly pairing the DEirlncRNA. The matrix was defined as 1 if the previous lncRNA had a higher expression than the latter; otherwise, it was defined as 0. A DEirlncRNA pair was considered as a valid pair when the number of pairs whose expression was set to 0 or 1 exceeded 20% of the overall number or was less than 80%.glmnet in the R software), 31 DEirlncRNA pairs showed high significance. Finally, 10 DEirlncRNA pairs were selected, and multivariate Cox regression analysis was used to construct a prognostic model. The following formula was used: risk score = (0.42791350 * RP11-613D13.8|RP11-576I22.2 expression) + (0.91712368 * HOXA11-AS|CDIPT-AS1 expression) +(0.44998528 * MIR663AHG|CDIPT-AS1 expression) + (−0.37327021 * PART1|HAND2-AS1 expression) + (0.62534486 * AP000695.6|RP11-492E3.2 expression) + (−0.81336299 * LA16c-325D7.1|RP11-884K10.6 expression) +(−0.76594801 * HOXC-AS1|LINC00460 expression) + (−0.24445084 * RP4-760C5.3|MIR663AHG expression) + (−0.47892575* RP11-1069G10.1|FLG-AS1 expression) + (−0.18018873 * CTD-2529O21.1|BVES-AS1 expression).Univariate Cox analysis was performed to evaluate the link between DEirlncRNA pairs and overall survival (OS) of GC samples in the training group. Eighty-one of DEirlncRNA pairs were significantly associated with OS. Through least absolute shrinkage and selection operator (LASSO) regression analysis  and receiver operating characteristic (ROC) curves were plotted at 1, 2, and 3 years using the “caret and proc package.In order to prove feasibility of randomly grouping stomach adenocarcinoma (STAD) patients from TCGA as the validation group, 10-fold cross validation was used to test algorithm accuracy of the prognosis model. Divide STAD dataset into ten parts, nine of which were used as training data and one as testing data in turn. The corresponding average AUC was obtained. The R packages used in these steps were Dseq2 identified the differentially expressed irlncRNA (DEirlncRNA) in the training cohort with log fold change |FC| > 2.0 and p-value < 0.05.Differential expression analysis between irlncRNAs performed using the R package rms package in R software and drew line segments with the scale on the same plane according to proportion for visualization. The R packages used in these steps were pHeatmap survival, rms, and ggupbr.The association between risk models and clinicopathological characteristics was analyzed using Chi-square tests. The Wilcoxon signed-rank test was used to assess differences in risk scores between groups for these clinicopathological characteristics. Univariate Cox regression analyses of risk scores and clinicopathological characteristics for the three cohorts were then used to verify whether the model could be used as an independent predictive factor for clinical prognosis. The results were demonstrated in a forest plot. Finally, we also performed nomograms based on multivariate regression analysis via the P < 0.05. The R ggplot2 software package was utilized in this procedure.Different methods, including CIBERSORT, MCPCOUNTER, XCELL, QUANTISEQ, TIMER, CIBERSORT-ABS, and EPIC were used to calculate the immune infiltration status of samples in the STAD dataset (TCGA) and explore the association between immune cell characteristics and risk. Wilcoxon signed-rank test was used to assess immune infiltrating cell types between the low and high-risk groups. Spearman correlation analysis was used to explore the association between immune infiltrating cells and risk score values. The threshold of significance was set at 50 of common administrating chemotherapeutic drugs in the STAD dataset (TCGA). The American Joint Committee on Cancer (AJCC) guidelines recommend chemotherapeutic drugs such as cisplatin, docetaxel, paclitaxel, mitomycin, and doxorubicin for GC treatment. Through the calculation of the Wilcoxon signed-rank test, the discrepancy in the IC50 between the high-risk and low-risk groups was obtained. By using “pRRophetic” and “ggplot2” of R package, the results were shown in the form of a box plot.To assess the model in the clinical treatment of gastric cancer, we measured the ICThe ggstatsplot′ R package was used to analyze the association between the expression level of genes related to ICIs and the novel model, and the results were visualized in the format of a violin plot.clusterProfiler. The threshold of significance was set at p < 0.05.Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GESA) pathway enrichment analyses were performed in R using the function of The study process is shown in Next, we annotated the data by gene transfer format (GTF) files from Ensembel. We performed differential expression analysis of lncRNAs between cancer tissues and normal tissues. Among them, 4,361 irlncRNAs were obtained  by the cP < 0.05). Further analysis of 81 DEirlncRNA pairs through LASSO regression analysis (p < 0.0001) by Kaplan-Meier analysis  of cisplatin  were  in all cohort's low- and high-risk groups. Ninety DEGs  were . Next, w05) were , cellula05) were , B cell 05) were , and B c05) were  were notGastric cancer is one of the most common malignant tumors and a highly heterogeneous disease , 7. A hiMALAT1 is involved in the gene expression process and post-transcriptional regulation of mRNA splicing process  through regulating PD-1/PD-L1 checkpoint, which suggests that targeting SNHG14 could be a promising way to improve the effectiveness of DLBCL immunotherapy  in bladder urothelial carcinoma  can be found in the article/TB and ZW designed the study and wrote and revised the manuscript. JX reviewed the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This work demonstrated a diselenide-containing protein hydrogel that could efficiently self-heal up to nearly 100% without compromising their mechanical properties under visible light at room temperature.Smart hydrogels are typical functional soft materials, but their functional and mechanical properties are compromised upon micro- or macro-mechanical damage. In contrast, hydrogels with self-healing properties overcome this limitation. Herein, a dual dynamic bind, cross-linked, self-healing protein hydrogel is prepared, based on Schiff base bonds and diselenide bonds. The Schiff base bond is a typical dynamic covalent bond and the diselenide bond is an emerging dynamic covalent bond with a visible light response, which gives the resulting hydrogel a dual response in visible light and a desirable self-healing ability. The diselenide-containing protein hydrogels were biocompatible due to the fact that their main component was protein. In addition, the hydrogels loaded with glucose oxidase (GOx) could be transformed into sols in glucose solution due to the sensitive response of the diselenide bonds to the generated hydrogen peroxide (H Self-healing hydrogel materials have attracted increasing interest due to their self-healing properties for a wide range of applications in biomedical systems ,2,3,4,5.Among these self-healing hydrogels based on dynamic covalent bonds, one of them employs light-induced dissociation and recombination of homolytic covalent bonds as the driving force for self-healing. The disulfide bond is one of the favorite light-driven dynamic covalent bonds, as it allows for dynamic metathesis under ultraviolet (UV) light ,23,24,25Herein, we presented a simple method for the construction of a dual dynamic bind, cross-linked, diselenide-containing protein hydrogel, based on Schiff base bonds and diselenide bonds . The pro2Se2 was for further use. 2-(Boc-amino)ethyl bromide (0.022 mol) in deoxygenated methanol (20 mL) was subsequently added to the above Na2Se2 solution and the reaction was stirred overnight at room temperature. The reaction stopped and the solvent was evaporated by vacuum. The product was washed 3 times with CH2Cl2 and deionized water. The organic layer was dried over anhydrous MgSO4. Boc-selenocystamine (Boc-SeCy) was purified by column chromatography for further use. Trifluoroacetic acid (TFA) (5 mL), with the obtained Boc-SeCy, was added to CH2Cl2 and the reaction was stirred for 4 h at room temperature. The solvent was evaporated under vacuum and ether (100 mL) was added to the system. SeCy was obtained by filtering and collecting the solids.Selenium powder (0.010 mol) and sodium borohydride (0.010 mol) were added to a three-necked flask (250 mL) and the whole system was then deoxygenated under a nitrogen atmosphere. Then, deoxygenated water (10 mL) was slowly added to the system and the reaction solution was stirred for 1 h at 40 °C until a clarified solution. After cooling down to room temperature, the obtained brownish-red solution NaThe protein BSA (210 mg) was dissolved in phosphate-buffered solution at pH 6.0 (2 mL) and mixed with 20% SeCy solution (100 μL). Then, 5% glutaraldehyde (364 μL) was homogenously mixed with the mixture. The diselenide-containing protein hydrogel was formed under visible light at the room temperature for 12 h. The heterochromatic protein hydrogels were prepared in the same way with the addition of rhodamine B and thioflavin T (0.5 mg/mL), respectively. The protein hydrogels with different selenium content were prepared in the same way by replacing part of the SeCy with hexanediamine. These chemically cross-linked hydrogels showed promising long-term stability without any apparent erosion observed.The scanning electron microscopy (SEM) image of the diselenide-containing protein hydrogel was recorded by a JEOL-JSM-6700F instrument . The samples were prepared for testing in the following way. Initially, little drops of the gelator mixture were placed on a silicon wafer and the mixture gradually developed a hydrogel on the wafer. The wafer containing the hydrogel was then immediately frozen in liquid nitrogen. At last, the sample of the frozen protein hydrogel was rapidly moved to a freeze-dryer and was freeze-dried for 24 h. The sample was covered with gold spray before SEM measurement.−1. The prepared hydrogels were placed for testing on the platform of a rheometer MCR 302. The rheological measurements were carried out at room temperature with a 25 mm diameter parallel plate in which the stress was manipulated by the rheometer. The storage modulus (G′) test and the loss modulus (G″) test were performed under the above-described conditions.The rheology of oscillations in diselenide-containing protein hydrogels was measured with frequency (oscillation frequency). In frequency scan tests, the frequency of the range was from 1 to 100 rad s−1.Tensile tests were carried out using an Instron-5940 tensile instrument  with a 100 N pressure measuring element. The diselenide-containing protein hydrogels were formed in strips of 3.0 mm × 4.0 mm × 12.0 mm in size. In tensile experiments, the self-repairing and tensile characteristics of the diselenide-containing protein hydrogels were measured at a rate of 20 mm min−2 at the sample position.In the hydrogel self-healing experiments, the strips of the protein hydrogels were severed in the middle and then butt-jointed, and a little buffer solution was added dropwise to the joint interface. The spliced protein hydrogels were left in visible light or in the absence of visible light for 16 h with no additional stress or heat. The center of the illumination was 20 cm from the hydrogels during illumination. The power of the lamp was 80 W, producing a light intensity of 9.3 mW cmFollowing a previously reported method, the self-healing properties of the hydrogel were assessed by the ratio of the maximum stress in the self-healing hydrogel to the maximum stress in the original hydrogel ,32.2O2 by oxidising glucose under mild conditions. Then, the prepared protein hydrogels containing GOx were placed in 0.2 M glucose solution and the transformation of the protein hydrogels was observed during different times.The protein hydrogels produced were sensitive and responsive to redox stimuli due to the presence of diselenide bonds. Based on the responsive properties of diselenide bonds, the hydrogels were degradable by enzymatic catalysis. We prepared the diselenide-containing protein hydrogels by adding GOx at 0.5/500 mg, which produced H1H NMR spectrum was recorded with Bruker AVANCE III 500  using a tetramethylsilane (TMS) proton signal as the internal standard. ESI-MS analysis was performed by the Thermo Finnigan LCQ Advantage Mass Spectrometer . The Boc-SeCy was firstly synthesized by the reaction of 2-(Boc-amino)ethyl bromide and Na2Se2. 1H NMR  δ: 5.04 , 3.48 , 3.01 , 1.45 3). ESI-MS: calculated m/z 448.0, found m/z 448.1, [M]+  δ: 3.44 , 3.19 . ESI-MS: calculated m/z 125.0, found m/z 125.0, [M + 2H]2+  experiments firstly showed that the SeCy had no effect on the secondary structure of GOx, ensuring that the catalytic oxidation of the GOx was not affected (In order to study the biocompatibility of the diselenide-containing protein hydrogels, MTT assays were performed according to the previous method . In vitraffected . Diselenaffected B. The reIn summary, we have succeeded in constructing a dual dynamic bind cross-linked protein hydrogel, based on Schiff base bonds and diselenide bonds. The diselenide-containing protein hydrogels have highly efficient self-healing properties in visible light due to the fact that diselenide bonds allow for dynamic bond exchange via radical reactions in visible light. Under visible light, the self-healing efficiency of the hydrogel by a double dynamic response reached up to nearly 100% with no compromise in mechanical properties at room temperature, which was much higher than the self-healing efficiency by a single dynamic response of Schiff base bonds at the same time. Thus, the use of visible light in protein hydrogel systems containing diselenide bonds can optimize the healing process, shorten the healing time and improve the healing effect. The diselenide-containing hydrogel system achieved gentle visible light remote recovery of fractured hydrogel materials and it is potentially promising in the recovery of systems that are hard to access and difficult to recover in long distances. Furthermore, the diselenide-containing hydrogel loaded with GOx was degradable under mild conditions by the addition of glucose in response to the stimulus of the diselenide bonds. In addition to the desirable recoverability and degradability, the remarkable biocompatibility makes the protein hydrogels a promising application in biomimetic materials and tissue engineering.


Obsessive-compulsive disorder is a mental disorder characterized by repetitive, unwanted thoughts and behavior due to abnormal neuronal corticostriatal-thalamocortical pathway and other neurochemical changes. Purmorphamine is a smoothened-sonic-hedgehog agonist that has a protective effect against many neurological diseases due to its role in maintaining functional connectivity during CNS development and its anti-inflammatory and antioxidant properties. As part of our current research, we investigated the neuroprotective effects of PUR against behavioral and neurochemical changes in 8-hydroxy-2-(di-n-propylamino)-tetralin-induced obsessive-compulsive disorder in rats. Additionally, the effect of PUR was compared with the standard drug for OCD, i.e., fluvoxamine. The intra-dorsal raphe-nucleus injection of 8-OH-DPAT in rats for seven days significantly showed OCD-like repetitive and compulsive behavior along with increased oxidative stress, inflammation, apoptosis, as well as neurotransmitter imbalance. These alterations were dose-dependently attenuated by long-term purmorphamine treatment at 5 mg/kg and 10 mg/kg i.p. In this study, we assessed the level of various neurochemical parameters in different biological samples, including brain homogenate, blood plasma, and CSF, to check the drug’s effect centrally and peripherally. These effects were comparable to the standard oral treatment withfluvoxamine at 10 mg/kg. However, when fluvoxamine was given in combination with purmorphamine, there was a more significant restoration of these alterations than the individualtreatmentswithfluvoxamine and purmorphamine. All the above findings demonstrate that the neuroprotective effect of purmorphamine in OCD can be strong evidence for developing a new therapeutic target for treating and managing OCD. Obsessive-compulsive disorder (OCD) is marked by recurrent, unusual as well as distressing thoughts (obsession) such as contamination, excessive washing of hands, hygiene, taboo thoughts, etc. Activities are performed to achieverelief from thesethoughts (compulsions), as patients believe that the completion of the ritual is the only preventive measure for the cause . OCD aff8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) is a 5-HT1A-receptor activator that induces preservative and obsessive behavior . It is aFor now, selective-serotonin-reuptake inhibitors (SSRI’s) are the first-line therapy for OCD . These aIn addition to the neurological system, the Shh-signaling pathway has been demonstrated to be critical in developing several organs, including the nervous system ,24. SoniSevere neuronal-cell-communication disruption, oxidative stress and glutamate excitotoxicity arise from its downregulation ,30. In aPurmorphamine (PUR) is a small chemical agonist for Smo receptors that is purine-derivative in nature . It trigIt has been shown that PUR has a protective effect in a variety of disorders, including Parkinson’s disease (PD) , ischemiWe receiveda total of 48 six-month-old adult Wistar rats weighing 250–300 g from the Central Animal House at the ISF College of Pharmacy in Moga, Punjab . Polyacrylic cages with a wire mesh top and soft bedding were utilized to hold the animals in the experiment. All animals were kept under conventional husbandry circumstances, which included a 12 h reverse light cycle, food and water (ad libitum), and a temperature of 23 ± 2 °C . Accordiv/v) Tween-80 ; Group5 8-OH-DPAT (8 μg/0.5 µL/2 min IDRN)+PUR (5 mg/kg i.p) [PUR5]; Group6 8-OH-DPAT (8 μg/0.5 µL/2 min IDRN)+PUR (10 mg/kg i.p) [PUR10]; Group7 8-OH-DPAT(8 μg/0.5 µL/2 min IDRN)+FLX (10 mg/kg p.o) [FLX10]; Group8 8-OH-DPAT(8 μg/0.5 µL/2 min IDRN)+PUR (10 mg/kg i.p.)+FLX (10 mg/kg p.o) [PUR10/FLX10].The experimental protocol lasted 42 days in total. From day 1 to day 7, an intra-dorsal raphe-nucleus injection of 8-OH-DPAT was given, and from day 8 to day 42, the animal grouping continuously received protocol drug PUR, i.p., and standard drug FLX, p.o. Animals were divided into eight groups using random selection. The current study was unblinded, and the experimenter was well-versed in animal care Group1 Vehicle Control; Group2 Sham control; Group3 PUR per se ; Group4 8-OH-DPAT (8 μg/0.5 µL/2 min IDRN) To begin, all of the animals in all of the groups were given the toxin in the morning for 1 to 7 days straight. Following that, from day 8 to day 42, the protocol drug purmorphamine was administered to PUR per se, PUR5, PUR10, and PUR10/FLX10. Fluvoxamine was administered to the animals in the groups FLX10 and PUR10/FLX10, which was the conventional treatment. The PUR10/FLX10 group received the fluvoxamine 30 min after the purmorphamine administration. On particular days, parameters such as spontaneous alternation behavior (SAB), compulsive checking behavior (CCB), marble-burying behavior (MBB), signal-attenuation behavior (SAT), and the forced-swim test (FST) were carried out in order to assess behavioral changes. On the 42nd day of the protocol schedule, all animals that had completed behavioral tests were decapitated, and their brains were separated for biochemical, inflammatory, and neurochemical analyses. An overview of the study’s protocol is summarized in w/v carboxymethyl cellulose were used to prevent dehydration [The 8-OH-DPAT-induced experimental model of OCD in the rat was carried out in accordance with the procedure established by Hjorth and Magnusson in 1988.Experimental rats were given intra-dorsal raphe-nucleus injections of 8-OH-DPAT for seven days, and the results were recorded. The study conducted by Hjorth and Magnusson in 1988 clearly demonstrated that 8-OH-DPAT reduced the levels of serotonin in several brain regions related toOCD. Rats were acclimated to laboratory conditions in order to avoid stress and trauma from occurring. Ketamine at a dose of 75 mg/kg was administered to anesthetize the rats . The anesthetized rat’s body was placed on a warm padded surface with the head secured by incision and ear bars in the stereotaxic frame . It was taken care that the bregma and lambda should be in the same position with proper alignment. Immediately after shaving, the rat’s scalp wascleaned with 70% ethanol to eliminate any debris and fur. A small incision with a scalpel was created in the mid-sagittal plane, the skin was retracted, and the skull was exposed to the bregma and lambda in order to determine the IDRN injection coordinates. Cotton buds were utilized to restrict bleeding, and carboxymethyl-cellulose eye drops containing 0.5 percent ydration . At anterior/posterior (AP) −7.8 mm, medial/lateral (ML) −3.1 mm and dorsal/ventral (DV) −7.5 mm with respect to the bregma, a hole was created under aseptic conditions and the cannula was implanted . To prevIn accordance with our newly developed experimental-protocol schedule for OCD, from the first to the seventh day of each week, 8-OH-DPAT  was administered through a 10 μL Hamilton microliter syringe attached with a 0.4 mm-outer-diameter hypodermic needle in the dorsal raphe nucleus for 2 min (0.5 μL/2 min). The rats were individually housed in polyacrylic cages with warm fabric and husk during the post-operative period, and careful attention was paid to them until spontaneous movement was restored. A constant temperature of 25 ± 3 °C was maintained in the operating room for approximately 2–3 h following anesthesia induction. After surgery, the animals were kept in their cages for 2–3 days with milk and glucose water to prevent any further physical harm. Their general state of health and dehydration were constantly monitored. Seven days after receiving a proper diet and water intake, the rats began to show symptoms of healing, such as regaining their ability to move freely.In the experimental protocol, weekly measurements of body weight were taken on days 1, 7, 14, 21, 28 and 42. Body weight was measured to see if there was a link between compulsive behavior and changes in food intake . Compulsive checking behavior was performed in order to evaluate ritualistic and environment-dependent compulsive behavior. It is characterized by repeated returns to one location in the environment . It was On the 10th, 20th, 30th, and 40th days of the protocol, each animal was examined for compulsive checking behavior. For ten minutes, the rats were released into an open field with four small plexiglass/glass boxes and allowed to move freely. Virtual cameras captured their every movement. Recorded behavior was used to calculate the frequency of visits and the duration of stops in each locale . The acquired values were utilized to determine the locale with the maximum cumulative visit frequency and the locale with the longest cumulative stop length. The checking behavior was defined in terms of the most-often-visited locale .In particular, four distinct types of trackingmetrics were employed to define compulsive behavior in OCD models: (a) checking frequency: total number of visits to the key locale; (b) checking-recurrence time: mean duration of return times to the key locale ; (c) stops before returning to the key locale: mean number of places visited between visits to the key locale; (d) checking length: total duration of stay at the key locale divided by the frequency of visits there. These metrics were examined by evaluating video footage of rats moving throughout a ten-minute period .On the12th, 22nd, 32nd, and 42nd days, the rats were tested in theFST to determine their depressive behavior. The rats were exposed to the tank for 15 min the first time, during the training session, and 24 h after the initial exposure, the rats were exposed fora second time for 5 min. For the rat, the 6 min testing period consisted of two minutes of habituation and four minutes of the actual test, which yielded the duration of immobility . n = 6 per group) were placed in transparent cages (41 cm × 36 cm × 10 cm) filled with 5 cm of sawdust. On top of the sawdust, in six rows of five, were thirty glass marbles, each measuring 1.5 cm in diameter, that were placed evenly. For 10 min, a 100 W bulb was used to illuminate the test area. After 10 min of exposure to the marbles, the rats were removed and the buried marbles were counted.As long as at least half of amarble wasburied, it was regarded as “buried” [Compulsion-like behavior was also measured using the MB test. As part of the protocol, it was performed on days 11, 21, 31, and 41. The rats . These arms were isolated by wooden doors that were manually opened. Small cups were placed at the corners of each goal arm. On the first day of training (day 1), the animals were given chocolate cake in their cages to help them overcome neophobia of novel food. The next day (day 2), the animals were given 20 min to get used to the T-maze, during which they were permitted to roam the entire space. They were then held in each goal arm for five minutes, at which point the reward was given to them the next day (day 3). The door was lifted, and they could choose either of the goal arms, which were baited with chocolate cake.A 15 s rest period was given to the animals after they finished eating the food. Cake was placed into an empty cup and the operation was repeated seven times per session. Animals’ decisions (left or right) and the time it took them to reach the target arm (up to 90 s) were recorded. The average number of goal-arm options was then calculated until an alternation occurred. As a result, rats with spontaneous alternation were awarded a score of 1, whereas preservers received a maximum score of 7 [In the signal-attenuation rat model of OCD, rats are trained to exhibit OCD behaviors by pressing a lever for food and then viewing a compound stimulus as a feedback cue. This distinguishes between compulsive and repetitive but not compulsive ,59.A lack of response-feedback systems or a lack of signaling that conditions have changed following the organism’s response can lead to obsession and compulsions. As a result, the only way to relax anxious behavior is to successfully complete an activity depicting ritualistic behavior. In OCD, signal attenuation leads to excessive lever pressing. This behavior is called compulsive leverpressing because it may be analogous to the excessive ritualistic and unreasonable behavior seen in OCD. The rats were tested usingthis model on the 11th, 21st, 31st, and 41st days of the protocol schedule.In our neuroscience lab, Mehan et al. modified and developed the instrument, successfully established and validated it with rodent behavior, and named it a rodent neurobehavioral analyzer for obsessive-compulsive disorder (OCD) and psychiatric disorders. The apparatus consisted of one operant chamber (45 cm × 26 cm × 27 cm), consisting of one lever (2 cm wide) and a food magazine for food delivery withdimensions (5.5 cm × 5.5 cm). Both the lever and food magazine werepositioned at 3 cm from each side of the wall with an inter-distance of 7 cm. A house light of 4 Watts was attached to the operant chamber. Signal was given in the form of light and sound. On providing thesignal magazine, the light was manually turned on along with the sound signal.Before the experiment began, the rats were handled for around two minutes a day for five days prior to the start of the trial. Handling and behavioral testing were both preceded by a 22 h food-restriction program. Immediately following the session, food was given to the rats in theircages. In the last three days, a tray containing 20–30 food pellets used in operant training was placed inside the cage and then removed. After at least two pellets were eaten by each rat, the tray was removed from the cage. Post-training: Day 1: HabituationRats were trained to collect food pellets from the magazine in the operant chamber. A magazine containing 4–5 food pellets was placed in front of the rats, who were given 5 min to consume all of the pellets from the magazine.Day 2–3: Magazine trainingRats were trained to collect food pellets from a food magazine. In each trial, a single food pellet was dropped into a food magazine through a food-dispenser tube simultaneous with the onset of the magazine light and sound. The stimulus was turned off after the rat head entered the food magazine or after 15 s with 30 s intervals between each trial. The number of completed trials (magazine entry during stimulus presentation) was observed within 30 min. Day 4–5: Lever-press trainingIn this training session, the delivery of food and the onset of the magazine light and sound were only initiatedwhen the rat pressed the lever. The stimulus was turned off after the rat head entered the food magazine or after 15 s from the lever press had elapsed. During the whole session (maximum 30 min) the rat should have completed 30 trials. Day 6: Signal attenuationIn this training, rats were exposed to a stimulus as in the lever-press training, but no food was delivered to the food magazine uponpressing the lever. The rat was trained until it had completed no more than 14 collected trials. Test day: The test was the same as that of the signal-attenuation stage. The number of completed excessive-lever-press trials were observed and recorded. The obsessive-compulsive-induced experimental rats showed more repeated trials than the normal and treated groups .w/v) ice-cold 0.1 M phosphate buffer (7.4) was used to homogenize the brain samples. Aliquots of the supernatant from the centrifuged homogenate were utilized for biochemical estimations after centrifugation at 10,000× g for 15 min [After decapitation, brains were extracted and cleaned with ice-cold isotonic saline solution on the 43rd day of the protocol schedule. Ten times . The animal was then scruffled with the thumb and forefinger of the non-dominant hand, and the skin around the eye was pulled tight. The medial canthus of the eye was punctured, and a capillary was introduced (30-degree angle to the nose). EDTA-containing Eppendorf tubes were used to collect the blood after puncturing the plexus or sinus. The plasma was then separated by centrifugation at 10,000×  at 4 °C . g for 10 min at 4 °C within 20 min of being collected. The supernatant was centrifuged and kept at −80 °C until further investigation [On day 43, the animals were sacrificedwith CHCl3, and CSF was collected according to the Rosenling et al., 2010 procedure. The rats’ heads were held in place using a device to display the arachnoid membrane. A small incision was madein the musculus trapezius pars descendens, followed by a horizontal incision. A maximum volume of 100 µL CSF was collected by directly inserting a 30 G butterfly-syringe needle into the cisterna magna via the arachnoid membrane. Each sample was centrifuged at a force of 2000× tigation .Smo-Shh levels in the rats’ brain-homogenate supernatant, blood plasma, and CSF were determined using an ELISA kit from Elabsciences in China. It is important to note that the data arereported as nM/g protein (brain homogenate) and ng/mL, respectively (blood plasma and CSF) .Caspase-3 levels were determined in both the brain homogenate and the blood plasma using an ELISA kit . The process was carried out in accordance with the manufacturer’s instructions. The competitive-enzyme-immunoassay technique was used in this assay, and the results arerepresented as ng/mg protein (brain homogenate) and ng/mL of sample (blood plasma) .The levels of Bcl-2 protein and Bax protein in the brain homogenate were determined using an ELISA kit  onthe samples. The process was carried out in accordance with the manufacturer’s specifications and instructions. The values arerepresented as ng/mg protein (brain homogenate) and ng/mL (blood plasma) . Using a diagnostic kit from Krishgen Diagnostics, India, the amount of TNF-α in the rats’ brain homogenate and blood plasma was determined in the experiments. All of the samples and reagents were produced in accordance with the instructions provided with the kit. The reaction mixture’s optical density was measured at 450 nmin microtiter plates. The data arepresented as pg/mg protein (brain homogenate), ng/mL, and ng/mg protein (brain homogenate) (blood plasma) ,68. A diagnostic kit from Krishgen Diagnostics, India, was used to test the concentration of IL-1β in the rats’ brain homogenate and blood plasma. According to the instructions on the kit, both samples and reagents were prepared as described. At a wavelength of 450 nm, the microtiter-plate reaction mixture’s optical density was measured. The data arepresented as pg/mg protein (brain homogenate), ng/mL, and ng/mg protein (brain homogenate) (blood plasma) .v/v (pH 4.5). Smooth separation wasensured by running the sample through the apparatus at a rate of 1 mL/min, and results are expressed in ng/mg protein [An electrochemical detector was used to measure the concentration of serotonin in the rats’ brain homogenate. There were two components to the mobile phase: buffer (sodium citrate) and acetonitrile (ACN) in the ratio of 87:13,  protein . It was possible to measure glutamate levels after derivatization with the compounds o-phthaldehyde/β-mercaptoethanol (OPA/β-ME). A quantitative examination of the tissue sample was carried out using an electrochemical detector (ECD) and high-performance liquid chromatography by HPLC-ECD Waters, USA, with the results represented as ng/mg protein .v/v) were used to buffer the mobile phase. NaH2HPO4 (NaH2HPO4) wasused in the buffer, which contained 10 mM citric acid, as well as 25 mM of EDTA (ethylene diamine tetraacetic acid). A voltage of +0.75 V and a sensitivity of 5–50 nA were used in the experiment. A flow rate of 0.8 mL/min was used to accomplish the separation. The samples (20 μL) were manually injected. A concentration of 0.2 M perchloric acid was used to homogenize rat-brain samples and centrifuge them for 5 min at 12,000× g. Results are expressed as ng/mg protein [High-performance liquid chromatography (HPLC) ECD Waters, USA and an electrochemical detector (ECD) were used to detect dopamine levels in the rat brain. Sodium citrate (pH 4.5) and acetonitrile . The assay mixture contained 0.05 mL supernatant, 3 mL 0.01 M sodium phosphate buffer of pH 8, 0.10 mL iodide acetylthiocholine, and 0.10 mL DTNB (Ellman reagent). Spectrophotometrically, the absorbance was immediately measured at 412 nm. The enzymatic activity of the supernatant is expressed as protein μM/mg . The lactate-dehydrogenase levelin the rats’ brain homogenate was measured using a diagnostic kit from Coral diagnostics, India, and is expressed as unit/mg protein .The spectrophotometric auto-oxidation of epinephrine was used to evaluate the SOD activity at pH 10.4. The reaction was started by adding 0.02 mL of epinephrine to the 0.2 mL of supernatant in the 0.8 mL of 50 mM glycine buffer. A 5 min spectrophotometric measurement of absorbance at 480 nm was performed. SOD activity is expressed asμM/mg protein . g for 15 min. Finally, 1 mL supernatant was added to 2.7 mL of phosphate buffer  and 0.02 mL of 5.5′ dithiobis-(2-nitrobenzoic acid). The 412 nm wavelength of a spectrophotometer was used to measure the yellow color’s intensity as soon as it was formed. In the supernatant, the glutathione content isexpressed asμM/mg protein [For the purpose of determining the concentration of reduced brain glutathione, 1 mL of supernatant was precipitated with 1 mL of 4% sulfosalicylic acid and cold digested at 4 °C for one hour. They were centrifuged at 1200×  protein . A colorimetric assay with Greiss reagent (0.1 percent N-(1-naphthyl) ethylenediamine dihydrochloride, 1-percent sulfanilamide, and 2.5% phosphoric acid) measures the accumulation of nitrite in the supernatant as a sign of nitric-oxide (NO) generation. Supernatant and Greiss reagents of equal volume were mixed together, and the combination was left to incubate in the dark at room temperature for 10 min and then spectrophotometrically tested for absorbance at 540 nm. The supernatant’s nitrite concentration was measured using the standard sodium-nitrite curve and is expressed as μM/mg protein .The level of malondialdehyde determines the lipid peroxidation in the rats’ brain homogenate. The amount of MDA wasmeasured by a spectrophotometer after its reaction with thiobarbituric acid at 532 nm. MDA concentration is expressed as nM/mg protein . p < 0.01 was statistically significant. The sample size was estimated after the data wereconfirmed to be normalized and the normality distribution was checked using the Kolmogorov–Smirnov test. GraphPad Prism version 5.03 for Windows was used to generate all statistical results . The mean and standard error of the mean areused to express the statistical data (SEM).Two-way ANOVA with Bonferroni post-hoc test and one-way ANOVA repeated measures with Tukey’s multi-comparison test were used to evaluate the data. It was determined that The body weight was measured weekly, i.e., at days 1, 7, 14, 21, 28, 35, and 42 of the protocol schedule. p < 0.01)when compared with the PUR5- and PUR10-treated rats. Additionally, FLX10 along with PUR10 showed a substantial restoration of body weight as compared to other drug-treatment groups such as PUR5, PUR10, and FLX10. PUR10 was shown to be more efficient in restoring the 8-OH-DPAT-induced reduction in body weight than PUR5, indicating the dose-dependent effect of PUR on restoring the body weight.The FLX10-treated rats showed a more considerable improvement in body weight [two-way ANOVA: F = 369.9, a. Decrease frequency of checking after purmorphamine treatmentThis parameter was testedon the protocol schedule’s 10th, 20th, 30th, and 40th days. Normal rats showed checking behavior, i.e., returning to a key locale when exploring. Adult rats treated with 8-OH-DPAT for seven days showed compulsive checking behavior. The rodents visited the key locale the most often compared to other locales, similar to compulsive checking in OCD patients. From day 8 to 42, the therapy medications (PUR and FLX) reduced compulsive checking at the key locale. The following four criteria were used to define the changes in behavior of all treatment groups.p < 0.01) than the PUR5- and PUR10-treated groups. Additionally, FLX10 and PUR10 had a more significant effect in reducing excessive returns to the key locale than the treatment withFLX10 alone. After 40 days of PUR10, the treated rats demonstrated more significant attenuation of an excessive number of visits when compared to PUR5 treatment of 8-OH OCD.b. Increased length of the check after purmorphamine treatmentp < 0.01) as compared to the PUR5- and PUR10-treated rats. Additionally, FLX10 and PUR10 had a more significant effect onincreasing the time that rats stayed at the key locale compared to the treatment withFLX10 alone. After 40 days, the PUR10-treatment group had a significantly increased length of check compared to the PUR5-treatment groupin 8-OH OCD (c. Increase recurrence time of checking after purmorphamine treatmentThe length of the check was defined as the mean time (in seconds) per visit that the rats stayed atthe key locale or spent time atthe key locale. Compared to the vehicle-control, sham-control, and PUR10-per se groups, the 8-OH-DPAT-treated rats spent less time at thekey locale. However, on the 20th day of the protocol schedule following drug treatment, the length of stay at the key locale began to rise compared with only the 8-OH-DPAT-induced experimental model of OCD in adult rats. At 30 days, the FLX10- and FLX10-and-PUR10-treated rat groups showed a significant increase in time spent at the key locale  = 15.59, 8-OH OCD b.c. Incp < 0.01) than the PUR5- and PUR10-treated groups. Additionally, FLX10 and PUR10 had a more significant effect onincreasing the time taken by the rat to return to the key locale compared to the treatment with FLX10 alone. After 40 days of PUR10 therapy, the rats spent more time at other locales and had a prolonged recurrence time of checking compared to PUR5 treatment of8-OH OCD.d. Increase the number of stops before returning to key locale after purmorphamine treatmentp < 0.01) than the PUR5- and PUR10-treated rats. Additionally, FLX10 and PUR10 had a more significant effect onincreasing the number of stops before returning to the key locale than the treatment with FLX10 alone. After 40 days, PUR10 treatment enhanced the number of stops in OCD-like rats compared to the PUR5 treatment  = 17.22, reatment d.As shown in p < 0.01) compared to the PUR5- and PUR10-treatment groups. Among the FLX-treated groups , FLX10 and PUR10 have a more significant effect on reducing immobility time than the treatment withFLX10 alone in 8-OHOCD. Additionally, PUR10 significantly reduced the immobility time in the FST compared to the PUR5-treated rats in a dosage-dependent mannerafter 32 days. These results suggest the antidepressant effect of FLX and PUR when given alone and a more significant improvement in the antidepressant effect when given together in 8-OH OCD. FLX10 administration, both alone and in combination with PUR10, resulted in a significant reduction in immobility time as  = 160, p < 0.01) when compared to treatments with PUR alone at two different doses (5 mg/kg and 10 mg/kg). FLX in combination with PUR led to agreater reduction in the number of buried marbles compared to treatment withFLX10 alone. After 41 days, PUR10 showed a more significant decrease in buried marbles than PUR5 in a dosage-dependent manner.p < 0.01)at day 30 than FLX10 treatment alone in 8-OH OCD.Treatment with PUR alone, as withFLX, reverses the 8-OH-DPAT-induced increase in the SAB score in a dose-dependent manner. On day 40 of the protocol schedule, PUR10 significantly reduced the SAB score compared to PUR5  = 25.14,  to PUR5 .p < 0.01)thanoral treatment with only FLX10 after 30 days. However, FLX10 treatment ofOCD-like rats lowered the ELP-C more than the PUR5 and PUR10 treatments in OCD-like rats. Furthermore, PUR10 demonstrated a dose-dependent anti-compulsive effect and was more effective than PUR5 atreducing the ELP-C in 8-OH OCD on day 41.The continuous excessive lever presses completed (ELP-C) signifies the compulsive and preservative behavior. p < 0.01), blood plasma  = 0.260, p < 0.01) and CSF  = 1.615, p < 0.01) as compared to the PUR5- and PUR10-treated groups. Additionally, compared to treatment with FLX10 alone, treatment with FLX10 and PUR10 combined had a more significant restoration of the reducedSmo-Shh level in 8-OH OCD. Furthermore, compared to PUR5, PUR10 treatment restored the lower levels of Smo-Shh in OCD like rats’ brain homogenate, blood plasma, and CSF. These results indicate that the level of Smo-Shh decreased in the OCD-induced experimental rats, then increased after the treatment with purmorphamine and the standard treatment of fluvoxamine  = 1.507, voxamine .p < 0.01) and Bax  = 0.189, p < 0.01) and decrease in the level of Bcl-2  = 1.989, p < 0.01) was restored after FLX and PUR treatment from day 8th to 42nd in 8-OH-DPAT-induced experimental model of OCD in adult rats. After seven days of 8-OH-DPAT administration, the levels of apoptotic markers such as Bax and Caspase-3 in the blood plasma and brain homogenate were relatively high compared to the vehicle-control, sham-control and PUR10-per se groups. In addition, the level of the anti-apoptotic marker Bcl-2 was shown to be significantly lower compared to the vehicle-control, sham-control, and PUR10-per se groups.This increase in the level of Caspase-3  = 1.783, p < 0.01), Bax = 0.863, p < 0.01) and Bcl-2  = 1.422, p < 0.01) levels in blood plasma among different groups. This reveals increased apoptosis in 8-OH-DPAT-induced OCD-like rats, reduced by purmorphamine and fluvoxamine administration, indicating anti-apoptotic activity  = 1.297, activity .p < 0.01)and IL-1β  = 2.254, p < 0.01)after 42 days of treatment. Among these treatment groups, those treated with FLX10, both alone and in combination with PUR10, indicate a more significant potential benefit in lowering the increased levels of pro-inflammatory cytokines than the PUR5- and PUR10-treated groups. Additionally, FLX10 and PUR10 treatment showeda considerably more reduced level of inflammatory markers than treatment with FLX10 alone in OCD rat groups. TNF-α-level evaluation by ELISAshowed that the repetitive intra-dorsal raphe-nucleus injection of 8-OH-DPAT triggers the release of TNF-α in the blood plasma and brain homogenate compared to the vehicle-control, normal-control, and PUR10-per se groups. The rat groups treated with FLX and PUR hadsignificantly decreased levels of TNF-α  = 0.315, p < 0.01)and IL-1β  = 0.756, p < 0.01)levels in the blood plasma among different groups. This study indicates that the intra-dorsal raphe-nucleus injection of 8-OH-DPAT causes an immunological response and the release of inflammatory cytokines in the rat brain, which is suppressed by purmorphamine treatment, which is similar to fluvoxamine treatment  = 1.773, reatment .p < 0.01) and dopamine  = 0.415, p < 0.01) while decreasing glutamate levels  = 0.417, p < 0.01) than the PUR5- and PUR10-treated groups. In the experimental study, serotonin and dopamine levels were shown to be lower in the brain homogenate of 8-OH-DPAT-treated rats; however, glutamate levels were found to be higher when compared to the vehicle-control, sham-control, and PUR10-per se groups. After 42 days, this neurotransmitter imbalance began to be restored in the FLX- and PUR-treated groups. FLX10, alone or in combination with PUR10, has a greater potential effect on raising serotonin  = 0.536, Compared tothe groups treated with FLX10 alone, FLX10 in combination with PUR10 had a more significant effect onrestoring neurotransmitter levels in the brain homogenate in8-OH OCD. Additionally, PUR10 treatment resulted in a significant increase in serotonin and dopamine and a decrease in glutamate in the brain homogenate of PUR10-treated rats compared to the PUR5-treated groups. These findings show that lower levels of serotonin and dopamine and elevated levels of glutamate cause an increase in repetitive and compulsive behavior in OCD-like rats, which is alleviated following a 42-day administration with PUR and the standard medication FLX .p < 0.01), MDA  = 0.937, p < 0.01), LDH  = 0.662, p < 0.01) and nitrite  = 0.463, p < 0.01) as compared to the PUR5- and PUR10-treatment groups. At the end of the experiment, the level of oxidative-stress markers was measured in the rats’ brain homogenate. Compared to the vehicle-control, sham-control, and PUR10-per se groups, the 8-OH-DPAT-treated groups had higher AChE, MDA, LDH, and nitrite levels and lower levels of reduced GSH and SOD. After PUR and FLX treatment from day 8 to day 42, the levels of these oxidative-stress markers becomerestored. FLX10treatment  showed a more significant effect ondecreasing the level of AChE  = 0.615, Additionally, FLX10 treatment alone and in combination with PUR10 showed a significant effect onincreasing GSH and SOD than PUR5 and PUR10treatment. Compared to the groups treated with FLX10 alone, FLX10 in combination with PUR10 had a more significant effect onrestoring oxidative-stress-marker levels in the brain homogenate in 8-OHOCD. PUR10 treatment showed a more significant decrease in AChE, MDA, LDH, and nitrite and increased the amount of decreased GSH and SOD in the brain homogenate compared to thePUR5-treated OCD-rat groups in a dose-dependent manner. This study found that FLX and theSmo-Shh agonist PUR protects against oxidative stress in 8-OH OCD .8-OH-DPATinduced compulsive checking behavior in rats, whereby rats were unable to resist returning to a key locale during exploration of the open field . This reSpontaneous alternation behavior in the T-maze is considered an appropriate model of OCD for assessing preservative behavior ,78,79. TIn the current findings, PUR’s anti-compulsive effect was measured by its potential to prevent 8-OH-DPAT-induced impairment of spontaneous alternation behavior compared to the standard OCD treatment fluvoxamine. PUR5 and PUR10 were also found to be effective in restoring alternation behavior but had a less-significant effect when compared with FLX.Marble-burying behavior was evaluated to measure characteristic repetitive compulsive-like behavior in OCD-like rats. It is a rodent’s defensive and unconditional behavior in which they repeatedly bury marbles that are not associated with any physical harm . During Signal attenuation is a model of OCD in which the rat excessively presses the lever not to collect the reward after signal-attenuation training . This beA forced-swim test was performed to assess the depressive effect in OCD as SSRIs are the principal therapeutic agent used in antidepressants . The depNeurochemical changes such as neurotransmitter imbalance, apoptosis, oxidative damage, and inflammation of neuronal cells are significant causes of the onset of compulsive-like behavior ,87. WhilOCD is primarily defined by abnormalities in the levels of various neurotransmitters in the brain. The most widely involved neurotransmitter in OCD is serotonin, which regulates various behavioral functions. Many clinical studies on OCD patients have also reported the significant effect of serotonin-reuptake inhibitors in treating OCD ,89,90,91Serotonin and dopamine deficiency impairs the motivational circuit related to the CSTC pathway and regulates behavioral characteristics ,94,95,96Our study found that the level of Smo-Shh was reduced after seven days of continuous injection with 8-OH-DPAT. This reduced level of Smo-Shh leads to increased neuroinflammation, oxidative stress, and apoptosis . Thus, POxidative stress is closely linked with anxiety and depression . This inIn summary, the current investigation found that PUR had a neuroprotective impact in alleviating 8-OH-DPAT-induced behavioral and neurochemical changes in OCD. These favorable benefits of PUR in attenuating behavioral alterations may be related to its ability to restore neurotransmitter levels and prevent brain damage via its anti-inflammatory and antioxidant characteristics. The considerable effect of PUR on OCD-like rats, when given in conjunction with FLX, suggests that it can be employed as a combination therapy in treating OCD more quickly and effectively. It is the first study to show that the Smo-Shh agonist PUR has a neuroprotective effect in OCD. Despite the lack of evidence regarding purmorphamine’s safety in humans, this chemical should be investigated further for the treatment of neurotoxicity linked with various infections, such as COVID-19.Limitations: In order to validate and confirm the mechanistic process behind the protective effect of PUR on treating OCD, various knock-in and knock-out studies of Smo-Shh genes are required. The primary drawback of this study is the use of whole-brain homogenate for neurochemical analysis and the absence of immunohistology and gross-pathology studies regarding the area-specific molecular mechanistic action of PUR. Along with this, gross-pathological, immunohistological, and interaction studies between FLX and PUR need to be performed in order to strengthen these studies’ findings and support the rationale for the clinical use of PUR, both alone and in combination with FLX, in preventing and managing OCD. Despite some limitations, this study signifies that the evaluation of serotonin, Smo-Shh in OCD patients, can be a possible biomarker in identifying OCD and can be prevented by early detection.Finally, this study found that purmorphamine has a neuroprotective effect in adult rats against the 8-OH-DPAT-induced experimental model of OCD. This is the first study to demonstrate that purmorphamine has a significant neuroprotective effect as a prospective medication for treating and managing OCD-like neurobehavioral abnormalities. The results of this study indicate that the Smo-Shh and serotonin assessments can be a possible candidate for the treatment of OCD-related neurocomplications.


Greater trochanteric pain syndrome (GTPS) is a musculoskeletal condition which can cause disability and reduce quality of life. However, limited evidence is available on the long-term outcomes of people with GTPS. Our aims were to determine the long-term prevalence of GTPS; to calculate the proportion of people with GTPS who had developed hip osteoarthritis (OA); and to determine the level of function and quality of life, 11-years after initial GTPS diagnosis.n = 24), asymptomatic control (ASC) group (n = 20)] were evaluated at baseline, 12-months and 11-years. At 11-years all participants completed the modified Harris Hip Score (mHHS), Oswestry Disability Index (ODI) and Assessment of Quality-of-Life questionnaire. At 11-year follow-up 20/24 GTPS and 19/20 ASC participants were clinically assessed for GTPS and hip OA, completed the 10 metre-walk-test, timed up and go, and hip abduction and external rotation strength testing.A prospective 11-year natural history study. Two groups : 10.19 , and 35.0% of GTPS participants were clinically diagnosed with hip OA compared to none of the ASC participants (p = 0.002), OR [95% CI]: 21.6, . GTPS participants reported more pain and disability than ASC participants via the ODI, mean difference [95% CI]: 6.1  but not the modified Harris Hip Score, mean difference [95% CI]: -3.3 . Both groups had similar levels of quality of life and measures of function.At 11-year follow-up 45.0% of GTPS participants had GTPS compared to 5.3% of ASC participants (GTPS is a chronic condition: people with GTPS at baseline had twice the odds of being clinically diagnosed with GTPS or hip OA than the control group at 11-years. Further, there appears to be a temporal relationship between GTPS and the development of hip OA. This finding highlights the need to identify effective treatments that address the underlying impairments associated with GTPS. Pain and function results varied depending on the assessment tools used. Between group differences in quality of life seen at baseline are not found at the 11-year follow-up. The small sample size means the results must be considered with caution.Level II Natural history Study.The online version contains supplementary material available at 10.1186/s12891-021-04935-w. Greater trochanteric pain syndrome (GTPS) causes pain on the lateral side of the hip with subsequent dysfunction which negatively impacts quality of life and reduces the ability to remain in full-time work, compared to healthy age matched controls . The undn = 164) reported at least 36% of people at 1-year and 29% of people at 5-years post diagnosis still had GTPS, with 24% self-reporting concurrent hip osteoarthritis (OA) : 10.19 . A large proportion of GTPS participants had gone on to develop hip OA, while none of the ASC had gone on to develop hip OA according to Altman’s criteria , OR [95%CI] = 21.6 . 18.2% of the GTPS had both GTPS and hip OA, while close to 80% of the ASC remained free of hip pain, Fig. At 11-year follow-up a larger proportion of GTPS participants had a clinical diagnosis of GTPS than the ASC participants, . This difference was not present at 12-months, or at 11-years. GTPS participants reported more disability on the mHHS and the ODI at baseline and 12-months, (p < 0.04) and on the ODI (but not the mHHS) at 11-years (p = 0.028), Table GTPS participants had a lower quality of life than ASC participants at baseline (p = 0.032). The GTPS group walked more slowly at baseline  than the ASC, and were slower on the TUG at the 11-year follow-up than the ASC, Table There was no group difference for hip abductor or external rotator strength at baseline or 12-month follow-up. The GTPS group had weaker hip abduction than the ASC at 11-years follow-up . By 11-years, only the ODI continued to have a  between group difference, noting that ODI has been found to measure a different construct than the subsequently developed condition specific VISA-G . The chaWe acknowledge several limitations within this project. Firstly, the 11-year follow-up diagnosis of hip OA was performed in the absence of an x-ray. The diagnosis of hip OA is more accurate when performed in combination with a radiographic examination , with veThis study demonstrates that many people with GTPS continue to experience hip pain (including due to GTPS) and appear to have a higher chance of developing hip OA after 11-years than a comparison group. These findings should be confirmed with a larger study. Clinicians involved in treating people with GTPS should be aware that over time their management strategies may need to change. Future studies should investigate biomechanical and somatosensory impairments and their effects on joint contact forces. Furthermore, investigations exploring effective long-term conservative management strategies are necessary to reduce the burden of GTPS, and the risk of developing hip OA.Additional file 1.Additional file 2.


Astaxanthin (ASX) is a potent lipophilic antioxidant derived from the natural pigment that gives marine animals their distinctive red-orange colour and confers protection from ultraviolet radiation. Self nano-emulsifying drug delivery systems (SNEDDS) have been successfully developed and evaluated to increase the skin penetration of ASX and target its antioxidant and anti-inflammatory potential to the epidermis and dermis. SNEDDS were prepared using a low-temperature spontaneous emulsification method, and their physical characteristics, stability, antioxidant activity, and skin penetration were characterized. Terpenes  were included in the SNEDDS formulations to evaluate their potential skin penetration enhancement. An HPLC assay was developed that allowed ASX recovery from skin tissues and quantification. All SNEDDS formulations had droplets in the 20 nm range, with low polydispersity. ASX stability over 28 days storage in light and dark conditions was improved and antioxidant activity was high. SNEDDS-L1 (no terpene) gave significantly increased ASX penetration to the stratum corneum (SC) and the epidermis-dermis-follicle region (E + D + F) compared to an ASX in oil solution and a commercial ASX facial serum product. The SNEDDS-containing D-limonene gave the highest ASX permeation enhancement, with 3.34- and 3.79-fold the amount in the SC and E + D + F, respectively, compared to a similar applied dose of ASX in oil. We concluded that SNEDDS provide an effective formulation strategy for enhanced skin penetration of a highly lipophilic molecule, and when applied to ASX, have the potential to provide topical formulations for UV protection, anti-aging, and inflammatory conditions of the skin. There is increasing consumer demand for effective, natural-based products to protect the skin from environmental assault and treat dermatological conditions related to skin aging, irritancy, and inflammation . AstaxanHaematococcus pluvialis is the main source for human applications including dietary supplements, cosmetics, and food. It has shown antiaging potential in an experimental model of oxidative stress [ASX has attracted interest due to its reported therapeutic effects in a range of health conditions . This ine stress . This suSkin aging has both cosmetic and health implications. The molecular and morphological changes that occur as the skin ages reduce its barrier function and protective role, and contribute to a range of skin symptoms including the formation or deepening of wrinkles, dyspigmentation, excessive dryness and pruritus, fragility, and difficulty in healing following skin injuries, skin sensitivity and irritation, and tumor incidence . A rangeASX  and evaluated the inclusion of terpene components on the skin permeation, stability, and antioxidant activity of ASX. SNEDDS are isotropic mixtures of an active compound in a combination of lipids, surfactants, and hydrophilic co-solvents/solubilizers that produce spontaneous ultrafine emulsions upon gentle agitation in the water phase  ,33,34. SWe have developed and characterized the physical characteristics, stability, skin permeation, and distribution, and antioxidant activity of SNEDDS composed of oil, non-ionic surfactants, and cosurfactants and incorporating ASX. We have also evaluated the inclusion of terpenes as potential skin penetration enhancers. To facilitate this study, we have developed a simple and accurate HPLC assay for ASX determination with suitable extraction protocols for quantifying ASX in skin layers.® EL (Polyoxyl 35 castor oil–CAS# 61791-12-6), D-limonene (CAS# 5989-27-5), geraniol (CAS# 106-24-1), farnesol (CAS# 4602-84-0), L-ascorbic acid (CAS# 50-81-7), potassium persulfate (CAS# 7727-21-1), and ABTS (Roche-Diagnostics GmbH) (CAS# 30931-67-0) were purchased from Sigma-Aldrich . Labrafil® M 1944 CS (Oleoyl polyoxyl-6 glycerides–CAS# 69071-70-1) and Transcutol® P (Ethoxydiglycol–CAS# 111-90-0) were kind gifts from Gattefossé . Astarism® facial serum was a gift from AstaREAL Co. Ltd., Minato-ku, Tokyo, Japan. Veet™ hair removal cream (containing potassium thioglycolate) was purchased from Reckitt Benckiser Pty Ltd. . HPLC-grade methanol and ethanol were from Thermo Fisher Scientific . HPLC-grade solvents (including acetone (ACT) and dichloromethane (DCM) were purchased from RCI Labscan Ltd.  distributed by Chem-Supply Pty Ltd., Gillman, Australia. Potassium chloride and sodium chloride were purchased from Chem-Supply Australia Pty Ltd. Potassium dihydrogen orthophosphate was purchased from Thermo Fisher Scientific . Di-sodium hydrogen orthophosphate anhydrous was purchased from Merck Pty Ltd . Deionised water was produced using a Milli-Q RC apparatus .Astaxanthin of analytical grade (CAS# 472-61-7) (SML0982) with a purity of 99.7%, KolliphorA stock standard solution of astaxanthin was prepared by weighing exactly 3.75 mg of astaxanthin into a 25 mL volumetric flask, dissolving the astaxanthin in a mixture (15 mL) of acetone and dichloromethane (50:50), and adding a mobile-phase solution to 25 mL. The solution was sonicated for 1 minute in a warm water bath at 60 °C, and allowed to equilibrate to ambient temperature for 15 min. Working solutions of individual standards were prepared by diluting the freshly prepared stock solution with the mobile phase to required concentration. ™ 1200 system , using a Jupiter C18 5 µm column, (150 mm × 4.6 mm) protected by a Security Guard Cartridge  both from Phenomenex , and isocratic flow of mobile phase (methanol:water:dichloromethane = 85:13:2) at 1 mL/min  and Kolliphor® EL (non-ionic surfactant), and Transcutol® P as a co-surfactant. Three terpenes  were added, as 5% w/w of the oil phase, to determine their ASX skin permeation enhancement capability within the SNEDDS formulation. The physical characteristics and ASX skin delivery of all formulations were characterized, including the droplet size, polydispersity index (PDI), viscosity, ASX solubility, stability, oxidative inhibition values, and amount of ASX retained in the skin (IVPT).SNEDDS were formulated with Labrafil® M 1944 CS, Kolliphor® EL, and Transcutol® P was determined from ASX solubility experiments. The SNEDDS were prepared based on the method of Hong et al. [The ratio of Labrafilg et al. , using aThe composition of the SNEDDS formulations is documented in ™ ZSP  with aqueous dilution 100-fold to avoid multiple scattering.The ASX SNEDDS were visually observed to describe the physical appearance , and viscosity , and refractive index  were determined. The pH measurement was determined using a pH meter  for all SNEDDS anhydrous formulations and after 100-fold dilution with deionized water. The droplet size and polydispersity index (PDI) were examined using a Zetasizer NanoRobustness to dilution was assessed in different volumes  using deionized water. This parameter provides a prediction of the potential for phase separation of a system generated by spontaneous emulsification. After the dilution process, the droplet size should remain uniform .The stability of ASX SNEDDS was determined over 4 weeks of storage at room temperature to provide a preliminary indication of the stability of ASX in the SNEDDS formulations, when exposed to or protected from light. Physical appearance and the ASX content were determined after storage at 22–25 °C in clear vials and protected from light in amber vials. ASX in oil solution was used as a control.Due to the lack of availability of human skin, newborn pig skin was used as a previously validated human skin surrogate ,43. Full®, UT58 series), as a measure of skin barrier integrity. Skin was included that provided a maximum resistance of 1 MΩ and minimum value of 50 kΩ [®, Kimberly-Clark Professional, Milsons Point, Australia). The receptor compartment was filled with a mixture of PBS and ethanol (7:3 v/v) to facilitate ASX solubility [®  was used to cover the donor and receptor cells to minimize evaporation and to facilitate the process of total replacement sampling. 0.5 g of ASX SNEDDS formulation, marketed ASX product or ASX in oil solution (control) was applied to the donor compartment as an infinite dose. Samples  were withdrawn for HPLC analysis and immediately replaced by pre-warmed receptor solution, over an 8 h period.The skin was defrosted at ambient temperature immediately before each experiment. Skin thickness was determined by digital vernier caliper  and was 400–600 µm. The skin was mounted in Franz diffusion cells with the stratum corneum (SC) side facing the donor. PBS solution was placed in the donor and receptor compartments and allowed to equilibrate at 35 °C prior to determining the electrical resistance  were applied on the skin surface with a D-Squame disc® applicator following the method of Davies et al. [After 8 h, the skin was washed to remove any remaining formulation (washing fluid retained), dried, and treated to separate SC and remaining skin tissue (epidermis/dermis). To obtain SC samples, D-Squames et al.  with modThe antioxidant activity of ASX was determined by the ABTS ) radical scavenging assay according to Chintong et al.  with min2S2O8, both in aqueous solution and equal quantities, were allowed to stand in the dark at room temperature for 16 h. ABTS working solution was then diluted with distilled water to obtain an absorbance of 0.70 ± 0.03 units at 734 nm using a UV–Vis spectrophotometer . Twenty microliters of different concentrations of the SNEDDS and ASX in ethanol 95% solution (200–1000 µg/mL) was mixed with 200 µL of the working ABTS solution. The absorbance of the mixture was measured after 6 min at wavelength 734 nm. Ascorbic acid and the marketed ASX topical product were compared as positive controls. Water and ethanol 95% were used as blanks. The scavenging rate of ABTS was calculated as:control, Asample, and Asample blank are the absorbance of the ABTS solution, ASX solution with ABTS, and ASX solution without ABTS, respectively.Briefly, 7 mM ABTS and 2 mM KFor the in vitro skin permeation study, the amount of ASX in the skin tissues was calculated from the HPLC analysis of extract solutions giving the amount of ASX retained in the SC (tape strips 2–10) and the amount of ASX present in the skin tissue .Mass balance of in vitro permeation experiments was determined by comparison of the initial applied amount of ASX with the combination of ASX in the wash, tape strips 1/2 and 3–10 samples, the remaining skin tissue extracts, and the receptor compartment.™ 9 software , using two-way ANOVA with Tukey post hoc test. Data were considered to be statistically significant if p < 0.05.All experiments were carried out in triplicate. The data are expressed as the mean ± SD  and the mean ± SEM . All data were analyzed using GraphPad PrismThe HPLC method provided a single ASX peak for the stock solution at 8.3 ± 0.1 min and good separation from any skin sample-related peaks see . The det® M 1944 CS (constituting the oil phase), Kolliphor® EL (non-ionic surfactant), and Transcutol® P as a co-surfactant. The optimal ratio was 1:4:1 (oil:surfactant:co-surfactant) providing the SNEDDS-L1 formulation. This ratio provided a transparent SNEDDS formulation that readily dispersed in water to produce a nanoemulsion with suitably small droplet size. The SNEDDS-L1 formulation was then adapted to include a terpene in the oil phase, providing SNEDDS-T1 (D-limonene), T2 (geraniol), and T3 (farnesol). The addition of 5% D-limonene, geraniol, and farnesol generated transparent nanoformulations with excellent clarity and similar physical characteristics to the SNEDDS-L1 formulation.The initial SNEDDS formulation was developed based on solubility studies of ASX with the three core excipients of LabrafilAll SNEDDS formulations were a darkish red-orange colour that formed a pale orange transparent nanoemulsion system when diluted 100-fold by deionized water A. The coThe droplet size and PDI were measured immediately after the preparation of each SNEDDS. In all cases the SNEDDS exhibited a monomodal distribution reflecting uniform size distribution, as shown for SNEDDS-L1 in ® EL used in formulations so that the surface charge was contributed only by co-surfactant.The zeta potential of all SNEDDS formulations, determined after dilution with distilled water, were −13.10 ± 0.52 to −12.40 ± 0.20 mV, and results are summarised in The robustness to dilution test showed minimal change in the droplet size and PDI of the SNEDDS formulation upon dilution with 50-, 100-, and 250-fold deionised water with the droplet size remaining approximately 20 nm for all formulations .No detectible ASX permeated through the skin to the receptor fluid from any of the SNEDDS, commercial formulation or ASX in oil (control) over the 8 h topical application period. In contrast, ASX was present in the SC and remaining skin comprised of the epidermis, dermis, and associated follicular regions . ® M 1944 CS, Kolliphor® EL, Transcutol® P, showed statistically significantly greater penetration of ASX into the SC compared to the marketed commercial product and control ASX in oil solution . The SNEDDS also deposited statistically significantly more ASX in the deeper skin (E + D + F) than the commercial product and the oil solution control . Overall, there was a 2.2-fold increase in ASX in the SC and a 3.6-fold increase in the amount of ASX in the (E + D + F) from SNEDDS–L1 compared to marketed ASX nanoformulation product.Three terpenes were added to the oil phase of the SNEDDS-L1 formulation to create SNEDDS-T1 (D-limonene), SNEDDS-T2 (geraniol), and SNEDDS-T3 (farnesol), and their effect on skin penetration determined . No ASX All SNEDDS-containing terpenes increased ASX permeation into the SC compared to ASX in oil solution control and marketed topical product. However, the ASX penetration was significantly greater only from the SNEDDS-T1 formulation. In comparison to the SNEDDS-L1, only the SNEDDS-T1 provided similar ASX penetration into the SC, with the SNEDDS-containing geraniol and farnesol showing lower but not significantly different ASX penetration. When considering ASX penetration into the deeper skin tissues (E + D + F), the terpene-containing SNEDDS all showed statistically significantly greater ASX penetration than the control and marketed topical product, with penetration in the rank order of SNEDDS-T1 > T2 > T3. In addition, all terpene-containing SNEDDS showed greater penetration than SNEDDS-L1, although the difference was significantly different only for the D-limonene formulation.The SNEDDS-L1 formulation was converted to an aqueous bearing nanoemulsion (designated L1-NE) by the addition of 50% water whilst maintaining the ASX concentration of the final nanoemulsion formulation. Addition of water significantly reduced the ASX penetration to the SC and (E + D + F) in comparison to the SNEDDS-L1 formulation . ASX penASX stability was determined for all SNEDDS formulations and a control solution of ASX in oil, over 1 month storage at 22–25 °C with light-protected (dark) and unprotected light conditions. ASX% remaining after 30 days was in the range of 86.67–93.33% for both dark and light conditions for the SNEDDS formulations A,B. The p < 0.05; The ABTS radical scavenging assay was performed to investigate the antioxidant activity of all SNEDDS formulations and the SNEDDS-based nanoemulsion (L1-NE), and compared to the marketed topical product and ascorbic acid . The ABTThe therapeutic potential of ASX has been investigated for a wide range of conditions and administration routes including oral and topical delivery . Topical® M 1944 CS (constituting the oil phase), Kolliphor® EL (non-ionic surfactant), and Transcutol® P as a co-surfactant (designated SNEDDS-L1) were successfully formulated and provided statistically significant enhancement of skin permeation of ASX compared to a marketed ASX facial serum formulation and ASX in oil solution  acid. It is a nonionic water-dispersible surfactant for lipid-based formulations that is commonly used as a co-emulsifier in topical formulations, and provides the oil phase for the current SNEDDS formulations. The selected surfactant, Kolliphor® EL (polyoxyl-35 hydrogenated castor oil), is a non-ionic solubilizer and emulsifying agent with hydrophile-lipophile balance (HLB) of 12. Its hydrophobic moiety is a combination of glycerol polyethylene glycol ricinoleate and fatty acid esters of polyethylene glycol, while the hydrophilic moiety is a combination of polyethylene glycols and glycerol ethoxylate [® P, a high purity grade of diethylene glycol monoethyl ether (DEGEE), was selected as the co-surfactant because of its properties as a hydroalcoholic solubilizer [Labrafilhoxylate . Transcuubilizer  and skinubilizer ,53. All ubilizer . The SNEDDS formulations provided excellent physical characteristics for skin application. The SNEDDS-L1 formulation showed small droplet size in the order of 18 nm, a negative zeta potential (12.40 ± 0.20 mV), and a viscosity (0.19 ± 0.01 Pas) that provided good spreadability and retention on the skin surface. Kaur and Ajitha reported that the delivery of fluvastatin increased when the droplet size and zeta potential are reduced and viscosity increased, suggesting that this was due to higher occlusivity on the skin . The droAll ASX-containing SNEDDS formulations showed good physical stability over 30 days and significantly improved the chemical stability of ASX in the formulation compared to ASX in oil solution, particularly when exposed to light . Whilst The SNEDDS-L1 formulation enhanced the penetration of ASX in the skin, providing a 2.2-fold increase and a 3.6-fold increase in the SC and (E + D + F), respectively compared to marketed ASX product. As ASX is highly lipophilic, it is expected to penetrate the lipid bilayer regions of the SC, but have poor partitioning to the more aqueous deeper epidermal and dermal tissues that constitute the target site for the ASX antioxidant and anti-inflammatory activity. When applied as the commercial formulation, the deposition of ASX in the E + D + F was lowest, followed by the oil solution. The SNEDDS-L1 formulation significantly increased ASX penetration to the target tissues compared to both the marketed topical product and a solution of ASX in oil. This demonstrates that the SNEDDS formulation components and their nanosystem structure has the ability to enhance skin penetration by a number of mechanisms. First, the oil can solubilize the lipophilic ASX thus presenting it to the skin in a form that can access the lipid domains in the SC. However, that would not explain the significant improvement in comparison to the ASX in oil solution. Second, the oil, surfactant, and co-surfactant components can modify the SC lipid structure to facilitate permeation within the SC and entry to the viable epidermal regions. Third, presenting the ASX as a nanosystem optimizes ASX presentation to the skin, and facilitates diffusion and partitioning within the epidermal layers. Indeed, it has been postulated that SNEDDS formulations act by taking up moisture from the skin surface to form an occlusive topical system that has a high thermodynamic driving force for skin delivery ,39,40. WTerpenes have demonstrated penetration enhancement activity by disruption of the intercellular lipid structure in the SC ,59 to inp < 0.05: Incorporation of terpenes increased ASX penetration to the deeper skin target site (E + D + F) compared to the SNEDDS-L1 formulation, with the following order of penetration SNEDDS-T1 > T2 > T3, although only the SNEDDS-T1 was significantly greater ASX penetration (We have demonstrated that SNEDDS are an effective formulation for enhanced skin delivery of the highly lipophilic ASX to the deeper skin tissues that constitute the target site for therapeutic antioxidant and anti-inflammatory activity. We have also shown that they provide the ASX in active form with high antioxidant activity  and help